The present invention relates in general to anticoagulant proteins, and in particular to novel recombinant blood coagulation inhibitors.
Tissue factor (TF) is generally considered to be the physiological trigger of the blood coagulation in normal hemostasis and in a variety of coagulopathic and thrombotic diseases. TF is an integral membrane protein that is normally present on the surface of certain extra-vascular cell types, but can also be induced to express on endothelium and monocytes upon stimulation [reviewed in (1)]. Based on studies in whole blood and re-constituted plasma systems (2-5), the key events of TF-initiated blood clotting can be schematically illustrated as in FIG. 1. Upon exposure, TF forms a complex with factor VII/VIIa present in the circulating blood. The resulting extrinsic tenase complex (TF/VIIa) initiates the clotting cascade by activating small amounts of factors IX and X on the TF-bearing cells/microparticles. The TF/VIIa-activated factors IXa and Xa play distinct roles in the subsequent coagulation reactions. In a complex with factor Va/V on TF-bearing membrane surface, factor Xa generates a small amount of thrombin that partially activates platelets, cleaves fibrinogen to form an initial clot, and converts factors V, VIII, and XI to their active forms. Subsequent to this initiation phase, propagation of thrombin generation begins. During the propagation phase, activated platelets provide an anionic membrane surface for the assembly of intrinsic tenase (VIIIa/IXa) and prothrombinase (Va/Xa) complexes, which very efficiently activate factor X and prothrombin, respectively, leading to explosive thrombin generation and consolidation of the fibrin-platelet plug. Three plasma anticoagulant systems regulate the clotting cascade, each acting at a different point in the cascade. Tissue factor pathway inhibitor (TFPI) influences the initiation phase by forming a TFPI-Xa inhibitory complex that inhibits TF/VIIa through feedback inhibition; antithrombin III (AT III) primarily exerts its effect by inhibiting free thrombin and Xa in the propagation phase; and activated protein C (APC) affects the duration of the propagation by proteolytically inactivating Va and VIIIa.
The availability of anionic phospholipid, chiefly phosphatidyl-L-serine (PS), is important for the assembly and expression of catalytic activities of the membrane-associated coagulation enzyme complexes (extrinsic tenase, intrinsic tenase, prothrombinase and XIa) that drive the initiation and propagation of the coagulation cascade. Plasma membrane phospholipids of mammalian cells are normally asymmetrically distributed, with PS being exclusively sequestered in the inner membrane leaflet (6). Because of PS sequestration, intact quiescent cells are normally not procoagulant. In circumstances of cell activation, cell injury, or in response to apoptotic stimuli, phospholipid asymmetry across the plasma membrane collapses, resulting in exposure of PS on the membrane surface and shedding of membrane “microparticles”. The exposure of PS allows assembly of enzyme/cofactor complexes and interaction with their substrates on the membrane surface, thereby enhancing the efficiency of the coagulation reactions (7-9). TF/VII(a) complex formed on intact cells is often cryptic in enzymatic activity towards its substrates. A many fold increase in TF/VIIa activity (de-encryption) is observed when PS becomes available on the membrane surface after cell disruption, treatments with various agents, or induction of apoptosis (10-14). The rate of factor X activation by TF reconstituted with vesicles composed of phosphatidylcholine (PC) alone is less than 5% of that observed with PS-PC vesicles (15). These observations suggest that concomitant expression of TF and exposure of PS on the membrane surface are important in the initiation of coagulation. In the processes of hemostasis/thrombosis, platelets are known to provide an anionic membrane surface for the assembly of intrinsic tenase (VIIIa/IXa) and prothrombinase (Va/Xa) (7,16). Upon platelet activation, PS rapidly appears on the platelet membrane surface. Interaction of factor VIIIa with the anionic lipid creates a Ca++-dependent high-affinity binding site for factor IXa, leading to the formation of the intrinsic tenase complex. Likewise, binding of factor Va to anionic lipid promotes Ca++-dependent binding of factor Xa, forming the prothrombinase complex. Factor XIa also depends on the PS-exposed membrane for efficient catalysis of the conversion of factor IX into factor IXa.
TFPI is a multivalent Kunitz-type inhibitor that regulates the initiation of the tissue factor pathway of coagulation in the human vascular system (17). TFPI inhibits factor Xa directly, and in a factor Xa-dependent manner, produces a feedback inhibition of TF/VIIa complex and thus dampens the protease cascade of the tissue factor pathway. Although TFPI is physiologically very important in the regulation of tissue factor pathway, its development for clinical antithrombotic therapy is currently limited by the large doses required for it to effectively interrupt vascular thrombosis (18-20).
Several other, naturally occurring Kunitz-type inhibitors that bind factors VIIa, IXa, Xa, and XIa of the tissue factor pathway have also been described. These include leech-derived Antistasin (ATS) (21), Tick Anticoagulant Peptide (TAP) (22), and two Ancylostoma caninum Anticoagulant Peptides (AcAP5 and AcAP6) (23) that inhibit factor Xa specifically; another Ancylostoma caninum Anticoagulant Peptide (AcAPc2) that inhibits VIIa (23); and a Kunitz-inhibitory domain of amyloid β-protein precursor (KAPP) that inhibits factors VIIa, IXa, Xa, and XIa (24-27). Using site-specific mutagenesis and phage display technology, two series of KAPP and aprotinin (bovine pancreatic trypsin inhibitor) homologs with very high affinity (sub-nanomolar Ki) toward different coagulation proteases (TF/VIa, Xa, XIa, and Kallikrein etc.) have been created (28-31). However, the anticoagulant potencies of these mutants are quite low in in vitro coagulation assays (tissue factor-initiated clotting and activated partial thromboplastin time). The aprotinin homologs also require very high doses to achieve antithrombotic effect in an in vivo vascular trauma model (31).